Historically, the cell culture experiments underlying biological understanding and drug candidate screening have been performed with homogeneous populations of cells on flat, stiff plastic substrates. Mounting evidence suggests that these stiff, static culture substrates only widen the gap between in vitro and in vivo assays. For many stem cells and multicellular constructs, 2D substrates are unable to recapitulate the tissue architecture and elicit biologically relevant responses. The extracellular matrix (ECM) surrounding cells in vivo is a complex, heterogeneous, and dynamic environment that exchanges biophysical and biochemical cues with cells. ECM mechanics and composition change as a function of time?during development, regeneration, repair, disease progression, and aging?and as a function of 3-dimensional location in organs. To increase the physiological relevance of in vitro cell culture experiments, researchers have developed biological and synthetic matrices that approximate the mechanics and water content of tissue. These ECM mimics represent a compromise between complexity and control. We will address two key challenges for 4D cell culture: reversible external control over the matrix, and independently tunable stiffness and stress relaxation. The objective of the proposed research is to develop OptoGels, a tunable platform for 4D cell culture technology. We have developed viscoelastic hydrogels that can be stiffened and softened by two different wavelengths of visible light. By installing a reversible photoswitch, azobenzene, adjacent to a dynamic covalent boronic ester linkage, we can control the binding constant for boronic ester formation with light. In preliminary experiments, we have achieved phototunable stiffnesses up to 9 kPa, spatiotemporal patterning of stiffness, and cytocompatibility. The Specific Aims of the proposed research are to (1) establish design parameters for OptoGels to maximize their versatility as cell culture materials, (2) benchmark mesenchymal stem cell and epithelial cell mechanobiology in OptoGels against literature data and commercial materials, and (3) engineer spatially controlled OptoGels that enhance the functional maturation of pluripotent stem cell-derived hepatocytes. Functional readouts and metrics that can be compared to established materials include YAP nuclear translocation, stem cell differentiation, cell spreading, cell migration, gene expression, and enzyme activity. The anticipated products of this research are a suite of OptoGel formulations with mechanical properties that can be tuned over physiologically relevant length and time scales. The long-term goal of this research is to develop user-defined cell culture materials that allow biomedical researchers to understand and control cellular processes relevant to human health.